Succinic acid is an intermediate in the tricarboxylic acid (TCA) cycle. Many bacteria are known to have natural ability to produce succinate as a fermentation product. The commercial demand for succinic acid is expanding. Succinic acid is used as a starting material in the manufacture of number of specialty chemicals such as 1,4-butanediol, tetrahydrofuran, γ-butryolactone and N-methylpyrrolidone.
A 2004 U.S. Department of Energy report entitled “Top value added chemicals from biomass” has identified succinic acid as one of the twelve building block chemicals that can be produced from renewable feedstock.
Currently, succinic acid is produced chemically from butane through maleic anhydride. The fermentative production of succinic acid from renewable feedstock is expected to become increasingly competitive with the rising oil price. Microbiological production of succinic acid has been reported with the rumen bacteria such as Actinobacillus succinogens, Anaerobiosprillum succiniproducens, Mannheimia succiniproducens, and Basfia succiniproducens (Scholten et al. (2009). Succinic acid production by these naturally occurring rumen bacteria depends on complex growth medium with expensive ingredients, which increases cost associated with the commercial production of succinic acid. In order to overcome these economic limitations associated with rumen bacteria, efforts have been made to engineer strains of Escherichia coli for commercial succinic acid production. For example, the E. coli strain K J122 for the production of succinic acid, described in by Jantama et at (2008a; 2008b) and in the International Patent Applications published under the Patent Cooperation Treaty Nos. WO 2008/115958 and WO 2010/115067, can grow on a minimal medium using glucose as the source of carbon.
Efforts have also been made to use eukaryotic microorganisms in the commercial fermentative production of succinic acid. International Patent Application Publication published on May 28, 2009 under Patent Cooperation Treaty with No. WO 2009/065777 describes a filamentous fungus genetically modified to produce a dicarboxylic acid. International Patent Application Publication published on May 28, 2009 under Patent Cooperation Treaty with No. WO 2009/065718 provides yeast strain with the genetic modification useful in the production of succinic acid.
In wild type E. coli grown under aerobic conditions, the carbon substrates such as glucose are fully oxidized. Further, the reduced cofactor pool (NADH) resulting from the complete oxidation of the substrate is oxidized through oxidative phosphorylation. On the other hand, under anaerobic condition the carbon substrates are only partially oxidized and fermentation products such as ethanol, lactate, acetate, formate, and succinate accumulate in the bacterial cells and fermentation broth. The efficiency of a genetically modified E. coli strain to produce succinic acid depends on carbon partition between various end products. Thus the efficiency of succinic acid production can be enhanced by blocking carbon entry into the other end products of fermentation. In addition to channeling the carbon flow into succinic acid, it is necessary to maintain the redox balance of the cell so that carbon flow from sugars to succinic acid occurs at high efficiency. During an anaerobic or microaerobic fermentation where the carbon source is one or more sugars, and where oxidative phosphorylation is absent or at a low level, cells need a biochemical mechanism for re-oxidizing the reduced compound NADH (and other similar reduced compounds such as FADH and NADPH) that is generated by the glycolytic pathway from sugar to pyruvate, For a cell to grow or live during an anaerobic or microaerobic fermentation, the net rates of oxidation of NADH to NAD and chemical reduction of NAD to NADH must be equal. Otherwise, the intracellular pool of either NADH or NAD will become depleted, and growth and metabolism will cease. A cell in which the net rates of oxidation of NADH to NAD and chemical reduction of NAD to NADH are equal is said to be “redox balanced”. Redox balance is more important to maintain under anaerobic or microaerobic conditions than under aerobic conditions, since when oxygen is present, any excess NADH can be oxidized by oxidative phosphorylation, and any deficit can be filled by running the oxidative TCA cycle.
In order to obtain commercially attractive yields of succinate, E. coli is grown anaerobically or microaerobically. Under microaerobic conditions, the fermentor can be exposed to the outside air through vents in the fermentor used for reagent exchange. In recent years, a method for producing succinate under aerobic condition has been described. U.S. Pat. No. 7,244,610 describes the aerobic succinate production using E. coli bacteria. When the bacteria are grown aerobically, the succinic acid production is achieved by means of blocking the normal operation of the oxidative tricarboxylic acid cycle through genetic manipulations. Thus under aerobic conditions, the deletion of the sdhA gene decreases oxidation of succinic acid into fumaric acid leading to the accumulation of succinic acid.
Two stage fermentations for producing succinate is also well known in the art. In the two stage fermentations, the bacterium is grown aerobically to achieve a certain cell mass followed by transfer to an anaerobic condition to start the succinic acid production. During the anaerobic production stage, the headspace of the fermentor is filled with carbon dioxide, hydrogen or a mixture of both gases. Thus in two stage fermentation, the bacterial growth phase is uncoupled from the succinate production phase.
Irrespective of the conditions used for the growth and succinic acid production, the succinic acid production in E. coli occurs through one of the three pathways illustrated in FIG. 1. As shown in FIG. 1, the central metabolic pathway starts with the glucose uptake. The wild type E. coli cells use an enzyme complex known as a phosphotransferase system (PTS) to transport the glucose from the medium into the cell. The PTS for glucose transport, for example, is a multi protein complex consisting of the PtsG, PtsI, PtsH and Crr proteins. The PTS system requires a molecule of phosphoenol pyruvate (PEP) for every molecule of glucose transported into the cell and yields a molecule of pyruvate for every molecule of PEP consumed. The glucose molecule transported into the cell is phosphorylated and ultimately two molecules of PEP are derived from each of the glucose molecule transported into the cell. Thus, of the two molecules of PEP that could be derived from a single glucose molecule, one molecule of PEP is consumed when the glucose uptake occurs through PTS and only one molecule of PEP is available for the central metabolic pathway to produce succinic acid. Thus the glucose uptake through PTS is an energy-intensive process that utilizes PEP substrate needed for succinic acid production and could reduce the efficiency of succinic acid production when using E. coli as a biocatalyst. This decrease in the efficiency of succinic acid production due to the PEP requirement at the level of glucose uptake is overcome by decreasing the activity of one or more of the genes such as ptsG, ptsI, ptsH and crr, which code for the polypeptides of the glucose PTS.
In the absence of a PTS, glucose and other sugars can be imported by an alternative sugar transporter located in the cytoplasmic membrane. One class of non-PTS transporters are the family of ATP binding cassette (ABC) transporters, which use ATP for energy. Another class of non-PTS transporters is the family of symporters that use an ion gradient (for example a proton gradient) for energy. A third class of non-PTS transporters is the family of facilitated diffusers, which requires no energy, but instead relies on a simple concentration gradient. For the non-PTS transporters, the imported sugar needs to be phosphorylated by a sugar kinase such as glucokinase, encoded by the glk gene. When the PTS is mutated, more of the PEP derived from glucose becomes available for biosynthesis, which in turn can increase the efficiency of production of certain chemicals such as succinate.
PEP can enter into the tricarboxylic acid cycle leading to the production of succinic acid by three different routes. In first pathway to succinate production, referred as the “reductive TCA” pathway for succinic acid production, PEP is carboxylated to oxaloacetic acid (OAA) by PEP carboxykinase or PEP carboxylase. The oxalaoacetic acid is reduced to malate which in turn is converted into fumarate. Fumarate is subsequently reduced to succinic acid. Thus through the reductive TCA pathway using PEP carboxykinase, succinic acid production is coupled with the oxidation of two molecules of NADH. This reductive pathway using PEP carboxykinase for succinate production is also reported to be present in the rumen bacteria Mannhaemia succiniproducens (Lee et al., 2005).
In the second pathway for succinic acid production, a molecule of PEP derived from glucose can be converted into pyruvate which in turn produces a molecule of acetyl-CoA. The acetyl-CoA thus produced can combine with the molecule of OAA to produce citrate which is isomerized to isocitrate. The isocitrate is decarboxylated to produce 2-ketoglutaric acid which is subjected to another decarboxylation reaction to yield succinyl-CoA, which is then converted to succinic acid. This second pathway for succinic acid production is referred as the “oxidative TCA” pathway for succinic acid production.
The third pathway for succinic acid production present in E. coli is known as the “glyoxylate shunt”, also known in some literature as the glyoxylate bypass. The glyoxylate shunt starts with isocitrate formed in the oxidative part of tricarboxylic acid cycle. The enzyme isocitrate lyase (encoded by aceA) cleaves isocitrate into succinate and glyoxylate. The glyoxylate combines with a molecule of acetyl-CoA, catalyzed by malate synthase (encoded by aceB), which results in the formation of a molecule of malate. Depending on the circumstances, the malate thus formed can be converted into fumarate, which in turn is reduced to succinate as in the reductive pathway for succinic acid production, or the malate thus formed can be oxidized to oxaloacetate, which can then combine with a second molecule of acetyl-CoA to enter the oxidative TCA pathway. This pathway, which has the net effect of converting two acetyl-CoA's and one NAD into one succinate and one NADH, is referred to as the “glyoxylate shunt”. The acetyl-CoA that feeds in to the glyoxylate shunt can come from pyruvate via pyruvate dehydrogenase, which also produces an NADH. Thus, when carbon flows from a sugar to succinate through the glyoxylate shunt, there is net production of NADH from NAD.
The maximum theoretical succinate yield in a redox balanced cell is 1.714 mol of succinate from 1 mol of glucose, which is equivalent to a mass yield of 1.12 grams of succinate for one gram of glucose consumed. This theoretical succinate yield is possible when the carbon flow through the central metabolic pathway is accompanied by appropriate redox balance. It has been reported that the maximum succinate production requires 71.4% of the carbon flow to OAA and 28.6% of the carbon flow to acetyl-CoA. (Vemuri et al., 2002a). Because the reductive TCA pathway to succinate requires a net input of two moles of NADH per mole glucose metabolized into succinate, and the glyoxylate shunt produces a net output of 5 moles of NADH per mole of glucose metabolized into succinate, it has been dogma in the prior art that redox balance for anaerobic or microaerobic succinate production from sugars in both bacteria and yeast is achieved by running a combination of the reductive TCA pathway and the glyoxylate shunt (Vemuri et al., 2002a; Sanchez et al., 2005; Wang et al., 2006; Martinez et al., 2010; Jansen et al., 2010). In fact, the flux models in these prior art disclosures do not even include the oxidative TCA pathway. This dogma was derived from earlier dogma that stated that a key enzyme in the oxidative TCA pathway, 2-ketoglutarate dehydrogenase “is virtually absent during anaerobic growth” (Cronan and Laporte, 1996).
In the prior art references cited above, succinate titers did not exceed 20 g/l, and the fermentation conditions required a rich medium. These titers are not high enough to be commercially attractive. Thus there has been a need for strains that produce succinate with increased efficiency. The strains of the present invention produce more than 20 g/l succinate in a minimal glucose medium.
Efforts have been made to enhance the carbon flow through the glyoxylate shunt for the purpose of improving the production of amino acids and chemicals. Two different approaches have been reported to be useful in enhancing the carbon flow through the glyoxylate shunt. In one approach, the enzymes involved in the operation of glyoxylate shunt are over expressed by means of replacing the native promoters with promoters constitutively active in E. coli. The two enzymes aceA (isocitrate lyase) and aceB (malate synthase) involved in the operation of glyoxylate shunt are in the aceBAK operon. U.S. Patent Application Publication No. US 2007/0015261 describes an E. coli strain expressing aceBAK operon under a constitutive promoter. In the second approach the glyoxylate shunt is activated by inactivating the iclR gene coding for a repressor of aceBAK operon. The inactivation of the iclR gene has been described in the U.S. Pat. Nos. 7,244,610 and 7,262,046.
In the present invention, the inventors have discovered that inactivation of the glyoxylate shunt had no negative effect, and inactivation of the glyoxylate shunt repressor (encoded by the iclR gene) had no positive effect on succinate production in the engineered E. coli succinate production strain KJ122. Deletion of the aceA gene coding for isocitrate lyase, the first enzyme in the glyoxylate shunt had no measurable effect on growth or succinic acid production in fermentors. Deletion of iclR also had no effect on growth or succinate production. Moreover, all attempts to delete any one of several genes encoding an enzyme of the oxidative TCA cycle, using well established methods, failed. Genes that could not be deleted in KJ122, include icd, encoding isocitrate dehydrogenase; gltA, encoding citrate synthase; sucD, encoding succinyl-CoA synthetase, and sdhD, encoding a subunit of succinate dehydrogenase. These results caused the present inventors to conclude that 1) the glyoxylate shunt is irrelevant and dispensable in KJ122, and 2) the oxidative TCA pathway is essential for growth and succinate production in KJ122.
Since the glyoxylate shunt springs from the oxidative part of the tricarboxylic acid cycle, these observations are useful in designing further genetic manipulations both on the oxidative and reductive parts of tricarboxylic acid cycle for the purpose of enhancing the production of succinic acid.